Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

All procedures followed were in accordance with the ethical standards of the institutions’ human research ethics committee. The study was approved by the Institutional Review Board at the Juntendo University Shizuoka Hospital, and written informed consent was waived because of the retrospective design.

1. Washing the slides

1. Prepare 10 µm of unstained formalin-fixed paraffin-embedded (FFPE) sections.
2. Place slides in a glass slide holder: use about 3–5 largest cross-sectional slides unless the tissue is minimal, and more slides are needed.
3. Fill the slide holder with xylene, and make sure that all tissue on the slide is submerged. Allow to sit for 15 min.
4. After 15 min, pour out the xylene with the slides held with a pipette tip so that the slides do not fall out.
5. Pour in more xylene to the same level as before. Allow to sit for another 15 min.
6. After 15 min, pour out the xylene again.
7. Fill the slide holder with 100% ethanol (EtOH), and make sure that all tissue on the slide is fully submerged. Allow to sit for 3 min.
8. Pour off the EtOH while carefully holding the slides. Refill to the same level with more EtOH. Allow to sit for 2 min.
9. Pour off the EtOH again and remove the slide. Carefully place them face up on a clean paper towel to dry. Allow to sit for 10 min.

2. Scraping the slides

1. Prepare digestion/lysis buffer with 650 mL of diethylpyrocarbonate (DEPC)-treated water, 100 mL of Ethylenediaminetetraacetic acid (EDTA), 50 ml of Tris hydrochloride (Tris-HCL) 2 M pH 8.8, and 200 mL of 10% sodium dodecyl sulfate (SDS) (see Table of Materials).
2. Fill a 1.5 mL single-use polypropylene tube with 80 µL of digestion/lysis buffer (see Table of Materials).
3. Put a clean pipette tip into the buffer.
4. Identify cancer tissues of the tumor area most suitable for macrodissection according to the appropriate H&E stained section.
   NOTE: The tumor area for macrodissection should be identified by preferably two qualified pathologists.
5. Macrodissect cancer tissue based on the marked H&E stained section.
   1. Take a clean razor blade and gently scrape the cancer tissue off the slide, trying to keep it in one piece so that it is easier to work with.
   2. Take the pipette tip out of single-use polypropylene tube containing buffer and use it to transfer the scrapped tissue into the buffer vial (see Table of Materials).
      NOTE: The wet tip should attract the tissue, which makes the transfer easier.
6. Repeat step 2.5 with the rest of the slides.
7. After all the tissue is in single-use polypropylene tube, use the tip to make sure the tissue is fully submerged and is not stuck to the wall of the tube.
8. Add 20 µL of a subtilisin-related serine protease to the vial and gently flick to mix (see Table of Materials).
9. Place the vial in a 55 °C heat block for at least 4 h or overnight. Make sure to slightly vortex after 2 h.

3. Bisulfite treatment

1. Use 45 µL of the digested tissue solution as the sample.
2. Perform the bisulfite treatment using reagents in a bisulfite conversion kit according to the manufacturer’s instructions (see Table of Materials).
   1. Add 5 µL of dilution buffer to the DNA sample and incubate at 37 °C for 15 min (see Table of Materials).
   2. While the samples are incubating, prepare the bisulfite conversion reagent by adding 750 µL of dH₂O and 210 µL of dilution buffer to one tube of CT conversion reagent (see Table of Materials). Mix the tubes by vortexing for 10 min.
   3. After the 15 min incubation, add 100 µL of the prepared CT conversion reagent to each sample and mix by inversion.
   4. Incubate the samples in the dark at 50 °C for 12 to 16 h.
   5. After the incubation, remove the samples and place on ice for 10 min.
   6. Add 400 µL of binding buffer and mix each sample by pipetting up and down (see Table of Materials). Load each sample into a spin column and place the column into a 2 mL collection tube (see Table of Materials).
   7. Centrifuge each sample at full speed (10,000 x g) for 1 min and discard the flow-through.
   8. Add 200 µL of wash buffer to each column and spin at full speed for 1 min, discarding the flow-through (see Table of Materials).
   9. Add 200 µL of desulfonation buffer to each column and allow the column to stand at room temperature for 15 min (see Table of Materials). After the incubation, spin the columns at full speed for 1 min and discard the flow-through.
   10. Add 200 µL of wash buffer to each column and spin at full speed for 1 min (see Table of Materials).
   11. Repeat this step one more time.
   12. Add 46 µL of dH₂O to each column and place each column in a new sterile 1.5 mL single-use polypropylene tube (see Table of Materials). Spin each tube for 2 min to elute the DNA.
3. Remove each spin column from the single-use polypropylene tube and discard (see Table of Materials). The DNA is now ready for the analysis.

4. Array platform for evaluation of DNA methylation at a CpG locus in the human genome

1. Assess the quality of DNA (quality check: QC) using FFPE QC assay on a real-time PCR amplification and detection instrument, with subsequent data analysis performed according to the manufacturers’ instructions (see Table of Materials).
   NOTE: Samples with ∆Cq values less than the recommended 5.0 are further processed¹⁴.
2. Analyze samples with an array platform for complex evaluation of DNA methylation to assess the methylation status of >450,000 CpG sites in the genome (see Table of Materials). Product information sheet, data sheet, and product files for the array platform are available¹⁵.
   NOTE: The assay is performed according to the manufacturer’s instructions for FFPE material¹⁴.
3. Calculate high and low locus methylation in a data analysis tool for an array platform as a β-value, with a range from 0 to 1, respectively (see Table of Materials). Use commercially available software (see Table of Materials) to calculate a β-value.
4. Import data generated on the array platform for complex evaluation of DNA methylation platform into the R software environment (R v.2.15.1) and process them using a tool for analyzing methylation arrays^16,17.
   NOTE: On the heatmap, which is generated using a data analysis tool, columns are ordered by unsupervised clustering, whereas the order of rows is based on the decreasing significance of the t statistic for differential methylation from top to bottom.
5. Divide the heatmap into high and low methylation groups using the first differentiator in the unsupervised clustering.
   NOTE: For validation with quantitative methylation-specific PCR (qMSP), choose genes based on a larger β-value in relation to CpG islands in the promoter region, and due to their suitability for primer and probe design for qMSP.

5. Quantitative Methylation-Specific PCR (qMSP)

1. Use the bisulfite-modified DNA from step 3.3 as a template for fluorescence-based real-time PCR in qMSP to evaluate methylation of the promoter region in each gene analysis.
2. Perform qMSP using a 96-well Real-Time PCR instrument (see Table of Materials).
   1. Check for the promoter methylation status of the target gene on the bisulfite-modified DNA using 200 nM forward primer, 200 nM reverse primer, and 80 nM probes. Prepare the master mix with 16.6 mM (NH₄)₂SO₄, 67 mM Tris pH 8.8, 10 mM β-mercaptoethanol, 10 nM fluorescein, 0.166 mM of each deoxynucleotide triphosphate, and 0.04 U/μl of DNA polymerase (see Table of Materials). The final reaction volume in each well for all assays is 25 μL.
   2. Perform cycling of qMSP as follows: 95 °C for 5 mins, followed by 55 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min.
      NOTE: Target gene should be chosen based on the criteria of having larger beta values, being related to CpG islands in the promoter region, and being suitable for primer and probe design for qMSP.
3. Use Human genomic treated with CpG Methylase (M.SssI) as a positive methylation control (see Table of Materials).
   NOTE: The final quantification of methylation is defined as the relative methylation value (RMV), and calculated as 2^–ΔΔCt for each methylation detection replicate compared to the mean Ct for β-Actin (ACTB)¹⁸. Primer and probe sequences are shown in Table 1. A Ct of 100 is used for undetected replicates, which gives a value of 2^–ΔΔCt close to zero. The following formula is used: mean 2^–ΔΔCt (RMV) = (2^{–ΔΔCt_replicate_1} + 2^{–ΔΔCt_replicate_2} + 2^{–ΔΔCt_replicate_3})/3¹⁸.